Field of the Invention
The present invention relates to the field of isotachophoresis (ITP), particularly for selective separation, detection, extraction, pre-concentration or quantitation of RNA, DNA, and/or other biological molecules.
Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual parts or methods used in the present invention may be described in greater detail in the materials discussed below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described.
Microfluidics has become an alternative to traditional techniques for biological and medical analysis and offers the use of small reagent volumes, fast analyses, and the potential for parallelization. 1 Polymerase chain reaction (PCR), capillary electrophoresis, 3 immunoassays, 4 and many other analytical techniques used in biology and medicine have been successfully miniaturized. However, sample preparation is often still a challenge and a limiting factor in the capability of many devices, so that most miniaturized systems have used prepurified, ideal samples as analyte. One important application is the purification of nucleic acids (NA) from complex biological samples, i.e., a complex mixture of macromolecules, which may contain small molecules as well. We here demonstrate a simple, fast, efficient, and sensitive technique for the purification of NA from whole blood which leverages the physicochemistry of isotachophoresis (ITP). The standard method for NA purification is based on solid phase extraction (SPE). For example, commonly used QIAGEN (Valencia, Calif.) purification columns rely on the adsorption of NA on silica membranes. 6 Extensive work by Landers and co-workers has shown successful microchip integration of SPE with application to purification of DNA7 and RNA8 and successful integration with on-chip PCR. While micro-SPE shows excellent efficiency and throughput, the process requires specialized materials and fabrication (e.g., micropillars or packing of silica beads). Further, the typical SPE protocol involves three successive steps (loading, washing, elution), requires bulk flow control, and uses a PCR inhibiting chemistry (e.g., chaotropic agents, organic solvents). Another example of SPE is the Quick Gene Mini-80, a nucleic acid extraction device by Fujifilm Life Sciences which uses pressurized filtration accompanied by washing and eluting steps to isolate nucleic acids.
ITP is a well-established separation and preconcentration technique. It leverages a heterogeneous buffer system to generate strong electric field gradients, allowing simultaneous focusing and separation of ionic species based on their effective electrophoretic mobilities. ITP has been marginally used as a sample purification method. For instance, Caslayska et al. (Caslayska, J.; Thormann, W. J., Chromatogr., A 1992, 594, 361-369) used ITP to simultaneously purify and isolate proteins. Kondratova et al (Kondratova, V. N.; Serd'uk, O. I.; Shelepov, V. P.; Lichtenstein, A., Biotechniques, 2005, 39, 695-699) concentrated and isolated extracellular DNA from blood plasma and urine by agarose gel ITP with applications to cancer diagnosis. This ITP isolation procedure yields DNA in an agarose gel slab which requires further purification steps prior to analysis. To our knowledge, ITP has never been applied to sample preparation from biological samples for analysis. The method in our invention is capable of accepting into the on-chip process a complex biological sample like whole blood added directly to the ITP well without pre-processing like centrifugation or filtration.
Presented below is an ITP-based purification method for extracting genomic DNA from a biological sample such as whole blood lysate as a sample. Previous work has been directed towards methods in which a complex sample such as this, containing a mixture of macromolecules, is pre-treated prior to ITP. In addition to separating Genomic DNA from cellular components such as membranes, organelles, proteins, and other nucleic acids.
In addition, the present methods relate to separation of small RNAs from other nucleic acids (sometimes referred to as polynucleic acids, as opposed to single nucleotides).
Small RNAs are involved in RNA interference (RNAi).
RNAi refers to the regulation of gene expression, in which small RNAs mediate gene silencing. In the RNAi process small RNAs are loaded onto Argonaute proteins at the core of an RNA-induced silencing complex (RISC), where these noncoding RNAs guide the sequence-specific silencing of transcripts through base-pairing interactions. The transcripts are typically messenger RNAs (mRNAs), which are cleaved or prevented from being translated by ribosomes, leading to their degradation. In humans at least 30% of the genes are thought to be regulated by miRNAs, which tune protein synthesis from thousands of genes. Further, miRNAs have recently been linked with common diseases.
miRNA expression profiling has been done using Northern blotting but this technique involves laborious, time-consuming procedures and lacks automation. The method of reverse transcription polymerase chain reaction (RT-PCR) is typically restricted to the quantitation of specifically lengthened miRNAs or pre-miRNAs, because the short length of miRNAs significantly limits the flexibility of primer design. Microarrays allow profiling miRNAs in a highly efficient parallel fashion, but this technique has encountered difficulties in reliably amplifying miRNAs without bias.
To overcome the obstacle of selective and sensitive quantitation devices for miRNA research, various techniques have been developed: a nanogapped microelectrode-based biosensor array; electrocatalytic nanoparticle tags and gold nanoparticle probes; and capillary electrophoresis with the sieving matrices of poly(ethylene oxide) or poly(vinyl pyrrolidone) for miRNAs, and poly(ethylene glycol) for general oligonucleotides applications. Compared to conventional capillary electrophoresis, microchip electrophoresis techniques offer considerably shorter analysis times, the ability to work with small sample volumes, and the opportunity of combination with additional on-chip assay steps. However, the loading of microchannels with gels remains challenging due to the high viscosity of crosslinked gels and consequential bubble formation.
Thus there exists a need in the field for fast and efficient method for extraction, isolation, preconcentration and quantitation of nucleic acids including small RNAs from complex biological samples like blood, blood lysate, cell culture, cell lysates, etc. For the present invention, we have extracted and purified nucleic acids from complex biological samples using ITP in microchannels and with PCR-friendly chemistries. We have extracted, isolated, preconcentrated and quantitated small RNAs from complex biological samples using ITP in microchannels loaded with a high efficiency sieving medium.